1. Volume 24, Issue 11, Pages 1898-1906 (November 2016)
A Phosphomimetic Mutation Stabilizes SOD1 and Rescues Cell Viability in the Context of an ALS-Associated Mutation 
James M. Fay, Cheng Zhu, Elizabeth A. Proctor, Yazhong Tao, Wenjun Cui, Hengming Ke, Nikolay V. Dokholyan 
Structure 
Volume 24, Issue 11, Pages (November 2016)
DOI: /j.str
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2. Structure 2016 24, 1898-1906DOI: (10.1016/j.str.2016.08.011)
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3. Figure 1
T2D-SOD1 and WT-SOD1 Have Similar Structures in Crystals and in Solution
(A) Overall ribbon diagram of the T2D-SOD1 homodimer (PDB: 5K02) with Zn2+ (silver) and Cu2+ (golden) shown in spheres. The superposition of T2D- (light green and light brown) and WT-SOD1 (purple, PDB: 1SPD) in one subunit indicates the minor structural change in the loop regions. The side chains of D2 and A4 are indicated as cyan sticks.
(B) Electron density map of the D2 residue and surrounding residues (residue numbers 22–29, 107–113) in one subunit.
(C) The CD spectra of T2D-SOD1 (cyan square) and WT-SOD1 (orange circle) indicate they both have β sheet structure in solution. See also Figures S1–S3.
Structure  , DOI: ( /j.str )
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4. Figure 2
T2D Stabilizes the Native State of SOD1 through Decreased Dimer Dissociation Rates
(A) Size-exclusion chromatograms showing the populations of native dimer (15.5 mL) and monomer (17.4 mL) for unmodified T2D-, T2D/A4V-, and WT-SOD1. Samples were taken after incubation at physiological conditions (30 μM SOD1 [pH 7.4]) and 37°C for 7 days.
(B) Size-exclusion chromatograms for glutathionylated T2D-, T2D/A4V-, and WT-SOD1.
(C–F) Dissociation of immobilized dimers was monitored by surface plasmon resonance for T2D-, T2D/A4V-, and WT-SOD1 (C) and glutathionylated species (D). Thermal denaturation curves of unmodified (E) and glutathionylated (F) SOD1 proteins indicate that the Tm of T2D-SOD1 is lower than that of WT-SOD1 in the unfolding of SOD1 monomers. Fitting to a two-state model is represented as lines. Size-exclusion chromatograms, dissociation profile, and thermal denaturation curves of A4V-SOD1 and GS-A4V-SOD1 have been reported (Redler et al., 2011). See also Figure S4.
Structure  , DOI: ( /j.str )
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5. Figure 3
T2D Mutation Reduces the Cytotoxic Effect of A4V in Motor Neuron-like Cells
(A) Transfection of NSC-34 cells with T2D-SOD1 and WT-SOD1 (negative control) led to a similar cell death ratio. A4V-SOD1 transfection (positive control) decreased cell viability and T2D/A4V-SOD1 rescued cells from A4V-SOD1-mediated cytotoxicity. Applied 3 days post-transfection, the red stain (propidium iodide) identifies dead cells, while the blue stain (Hoechst) identifies all cell populations.
(B) Average cell death rates are measured as the percentage of red-stained cells: WT 5.4%, T2D 5.6%, A4V 20.7%, and T2D/A4V 6.0%. Error bar represents the SEM, n = 3.
(C) Levels of the apoptotic marker caspase 3 in NSC-34 cells demonstrated by western blot confirm the protection role of the T2D mutation, because T2D/A4V leads to reduced expression of caspase 3. The normalized expression levels are: WT 100%, T2D 89%, A4V 377%, and T2D/A4V 135%.
Structure  , DOI: ( /j.str )
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6. Figure 4
DMD Simulation Suggests the Key Interface Interactions Are Disrupted in A4V-SOD1, in Contrast to WT-, T2D-, and T2D/A4V-SOD1
The flexibility of the protein backbone is measured as root-mean-square fluctuation (RMSF; red, 3.5 Å; blue, 0.5 Å) and the results are an average of ten DMD trajectories: WT-SOD1 (A), A4V-SOD1 (B), T2D-SOD1 (C), and T2D/A4V-SOD1 (D). The hydrophobic contact between V148-V148 is indicated as a green dashed line. The zinc-binding loop (Zn loop) that allosterically modulates the C4F6 epitope is indicated in the WT-SOD1 structure. See also Figures S5 and S6.
Structure  , DOI: ( /j.str )
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7. Figure 5
A Proposed Mechanism of the Regulation of Dimer-Monomer Equilibrium in SOD1 by Post-Translational Modifications
T2-Phosphorylation and C-111-glutathionylation may regulate the dimer-monomer equilibrium and have opposing effects on the stability of native SOD1 dimers. The T2D mutation (or T2-phosphorylation) inhibits the dimer dissociation (decreased koff), whereas C111-glutathionylation inhibits the association of monomers (decreased kon).
Structure  , DOI: ( /j.str )
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